So Many Columns! How Do I Choose? Daron Decker Chromatography Technical Specialist

So Many Columns! How Do I Choose? Daron Decker Chromatography Technical Specialist

GC Columns Wall Coated Open Tubulars Liquid phase coated capillaries Internal Diameter 0.05 0.53mm Length 5m 100m Porous Layer Open Tubulars Adsorbent coated capillaries Internal Diameter 0.25 0.53mm Length 5 60m Packed Columns Tubing packed with coated packings or porous particles Internal Diameter 2 4mm Length 0.5 10m

WCOT Column Types Agilent J&W has over 50 different stationary phase offerings

FactorFour TM Phases VF-1ms, VF-5ms, VF-5ht, VF-5ht UltiMetal VF-17ms, VF-17ms for PAH, VF-35ms, VF-200ms,VF-Xms, VF-23ms, VF-624ms, VF-DA, VF-1301ms, VF-Pesticides, VF-1701ms, VF-WAXms

20+ Different Specialty Phases Specialty phases are columns that are optimized to perform a specialized GC analysis. Column Typical Application DB-624 EPA and USP volatiles DB-VRX volatiles analysis HP-VOC volatiles analysis DB-502.2 EPA Method 502.2 DB-5.625 EPA semi-volatiles analysis DB-608 EPA Method 608 DB-1701P EPA pesticides analysis DB-MTBE total petroleum hydrocarbon (TPH) HP-PONA petroleum hydrocarbon analysis DB-HT SimDis hi-temp simulated distillation DB-ALC1 & ALC2 blood alcohol analysis HP-88 fatty acid methyl ester (FAME)

Select TM Column Examples Environmental applications CP-Sil 88 for dioxins, Select mineral oil, CP-Select 624 CB Chiral applications CP-Chirasil Val, CP-Chirasil-DEX CB Chemical applications CP-Volamine, CP-Select CB for MTBE, CP-PONA C8, CP-Propox, Select Silanes, CP-SimDist UltiMetal TM, CP-Lowox TM Food and Beverage applications CB-Carbowax 400, Select FAME, CP-Sil 88 for FAME, CP-FFAP

PLOT Column Types PLOT columns are primarily, but not exclusively, used for the analysis of gases and low boiling point solutes (i.e., boiling point of solute is at or below room temperature). Agilent J&W PLOT columns begin with the designation of GS (Gas Solid) or HP-PLOT followed by a specific name 10 stationary phases GS-OxyPLOT GS-Alumina HP-PLOT Al2O3 M HP-PLOT Al2O3 S HP-PLOT Al2O3 KCl GS-OxyPLOT: oxygenates HP-PLOT Molesieve: O2, N2, CO, Methane HP-PLOT Alumina and GS-Alumina: complex hydrocarbon gas matrices, ethylene and propylene purity, 1,4-butadiene HP-PLOT Q: freons, sulfides HP-PLOT U: C1 to C7 hydrocarbons, CO2, Polar Hydrocarbons GS-GasPro: freons, sulfurs, inorganic gases GS-CarbonPLOT: inorganic and organic gases HP-PLOT MoleSieve GS-CarbonPLOT HP-PLOT Q HP-PLOT U GS-GasPro

PLOT GC Columns from Varian Line Porous Polymers CP-PoraBOND Q CP-PoraBOND U CP-PoraPLOT Q CP-PoraPLOT U CP-PoraPLOT S CP-PoraPLOT Q-HT CP-PoraPLOT amines Zeolites Molsieve 5A Alumina KCL Na 2 SO4 MAPD Multi Layer CP-Lowox Porous Silica SilicaPLOT Graphatised Carbon CP-CarboPLOT P7 CP-CarboBOND Select TM permanent gases Molsieve 13x

Typical Capillary Column Polyimide Coating Fused Silica Stationary Phase Expanded view of capillary tubing

Distribution Constant (K C ) Mobile Phase Stationary Phase K C = conc. of solute in stationary phase conc. of solute in mobile phase K C formerly written as K D

K C and Retention Fused Silica Tubing Stationary Phase Gas Flow Stationary Phase K c => Large retention K c => Small retention

K C and Peak Width Time of Elution Fused Silica Tubing Stationary Phase Gas Flow Stationary Phase K c => Large K c => Small

Resolution Getting One Peak Away From Another 1 3 2 4

Separation vs. Resolution Separation: time between peaks Resolution: time between the peaks while considering peak widths

Column Selection Principles 1. Choose a stationary phase 2. Column Diameter 3. Film Thickness 4. Column Length

Resolution N k Rs = 4 k 1 a 1 a Efficiency Retention Selectivity N = (gas, L, r c ) k = (T, d f, r c ) a = (T, phase) L = Length r c = column radius d f = film thickness T = temperature

Selectivity Relative spacing of the chromatographic peaks The result of all non-polar, polarizable and polar interactions that cause a stationary phase to be more or less retentive to one analyte than another

Separation Factor (a) a = k 2 k1 co-elution: a = 1 k 2 = retention factor of 2nd peak k 1 = retention factor of 1st peak

Optimizing Selectivity Match analyte polarity to stationary phase polarity -like dissolves like(oil and water don t mix) Take advantage of unique interactions between analyte and stationary phase functional groups

Polarity Nonpolar Molecules - generally composed of only carbon and hydrogen and exhibit no dipole moment (Straight-chained hydrocarbons (nalkanes)) Polar Molecules - primarily composed of carbon and hydrogen but also contain atoms of nitrogen, oxygen, phosphorus, sulfur, or a halogen (Alcohols, amines, thiols, ketones, nitriles, organo-halides, etc. Includes dipole-dipole interactions and H-bonding) Polarizable Molecules - primarily composed of carbon and hydrogen, but also contain unsaturated bonds (Alkenes, alkynes and aromatic compounds)

Selectivity 100% PEG 4 1 6 Strong Dispersion Strong Dipole Moderate H Bonding 2 Compounds Polar Aromatic Hydrogen Bonding Dipole Toluene no yes no induced Hexanol yes no yes yes Phenol yes yes yes yes Decane no no no no Naphthalene no yes no induced Dodecane no no no no 5 3 100% Methyl 0 2 4 6 8 10 12 14 16 1 2 3 4 5 6 Strong Dispersion No Dipole No H Bonding 1. Toluene 2. Hexanol 3. Phenol 4. Decane (C10) 5. Naphthalene 6. Dodecane (C12) 0 2 4 6 8 10 12 14 16

Selectivity is important but not everything Temperature limits will play a role as well. Bleed and Inertness can be critical factors in column selection.

Polarity vs Stability/Temperature Range Polarity Stability Temperature Range

Benefits of Low Bleed Phases PAH Sensitivity Using DB-35MS 1 2 3 4 5 6 7 8 Commercially Available 35% phenyl column DB-35MS 9 10 11 12 13 14 Benzo[ghi]perylene S/N = 15 Benzo[ghi]perylene S/N = 120 15 16 1. Naphthalene 2. Acenaphthylene 3. Acenaphthene 4. Fluorene 5. Phenanthrene 6. Anthracene 7. Fluoranthene 8. Pyrene 9. Benz[a]anthracene 10. Chrysene 11. Benzo[b]fluoranthene 12. Benzo[k]fluoranthene 13. Benzo[a]pyrene 14. Indeno[1,2,3,- c,d]anthracene 15. Dibenz[a,h]anthracene 16. Benzo[g,h,i]perylene 5 10 15 20 25 min. Columns: 30 m x 0.32 mm x 0.35 um. Carrier: H2, constant flow, 5 psi at 100 o C. Injector: 275 o C, splitless, 1 ul, 0.5-5ppm. Oven: 100 o C to 250 o C (5 min.) at 15 o C/min.,; then to 320 o C (10 min.) at 7.5 o C/min. Detector: FID, 320 o C.

Poor Inertness Can Cause Poor Peak Shape Sample: 0.5 ng on column loading of Short Mix Components with ISTD Column: Brand R 20m 0.18mm 0.18µm Carrier: Helium 37cm/sec, Ramped flow; 0.7ml/min (0.1min) to 1.3ml/min (15ml/min 2 ) Oven: 35 o C (2.5 min) to 80 o C (40 o C/min), 15 o C/min to 200 o C, 8 o C/min to 275 o C (2 min) Injection: 0.5µl, Splitless, 280 o C, purge flow 30ml/min at 0.75 min MSD: Transfer Line 290 o C, Source 300 o C, Quad 180 o C 3 5 6 12 100000 50000 2 10 11 14 15 17 16 18 1. n-nitrosodimethylamine 2. Aniline 3. 1,4-Dichlorobenzene-d4 4. Benzoic Acid 5. Naphthalene-d8 6. Acenaphthene-d10 7. 2,4-Dinitrophenol 8. 4-Nitrophenol 9. 2-Me-4,6-dinitrophenol 10. 4-Aminobiphenyl 11. Pentachlorophenol 12. Phenanthrene-d10 13. Benzidine 14. Chrysene-d12 15. 3,3 -Dichlorobenzidine 16. Benzo[b]fluoranthene 17. Benzo[k]fluoranthene 18. Perylene-d12 Abundance 13 1 4 8 7 9 0 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Time (min) Agilent Restricted Month ##, 200X

Agilent J&W HP-5ms Ultra Inert Delivers Excellent Peak Shapes Sample: 0.5 ng on column loading of Short Mix components with ISTD Column: Agilent J &W HP-5ms Ultra Inert 20m 0.18mm 0.18µm Carrier: Helium 37cm/sec, Ramped flow; 0.7ml/min (0.1min) to 1.3ml/min (15ml/min 2 ) Oven: 35 o C (2.5 min) to 80 o C (40 o C/min), 15 o C/min to 200 o C, 8 o C/min to 275 o C (2 min) Injection: 0.5µl, Splitless, 280 o C, purge flow 30ml/min at 0.75 min MSD: Transfer Line 290 o C, Source 300 o C, Quad 180 o C 12 100000 5 6 50000 2 3 10 11 14 15 16 17 18 1. n-nitrosodimethylamine 2. Aniline 3. 1,4-Dichlorobenzene-d4 4. Benzoic Acid 5. Naphthalene-d8 6. Acenaphthene-d10 7. 2,4-Dinitrophenol 8. 4-Nitrophenol 9. 2-Me-4,6-dinitrophenol 10. 4-Aminobiphenyl 11. Pentachlorophenol 12. Phenanthrene-d10 13. Benzidine 14. Chrysene-d12 15. 3,3 -Dichlorobenzidine 16. Benzo[b]fluoranthene 17. Benzo[k]fluoranthene 18. Perylene-d12 13 Abundance 4 8 7 9 1 0 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Time (min) Agilent Restricted Month ##, 200X

Compare the Difference 12 100000 5 6 3 10 17 14 16 2 11 18 50000 15 13 Abundance 4 7 8 9 1 0 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Time (min) 100000 2 3 5 6 10 12 14 17 16 18 50000 11 15 13 Abundance 1 7 8 9 4 0 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Time (min)

Agilent J&W Ultra Inert GC columns Highly Inert Less absorption Improved quantification low levels Agilent s stringent QC testing Improved linearity Better peak shape Column reproducibility Better accuracy Improved separation More assurance Less re-runs DB selectivity Easy method change Less revalidation High Efficiency format Fast GC methods Higher productivity

Stationary Phase Selection Existing information Critical separations Selectivity/Polarity Inertness and Temperature limits Application designed Examples: DB-VRX, DB-MTBE, DB-TPH, DB-ALC1, DB-ALC2, DB-HTSimDis, DB-Dioxin, HP-VOC, and now Select TM phases Choose the column phase that gives the best separation but not at the cost of robustness or ruggedness.

Resolution N k Rs = 4 k 1 a 1 a Efficiency Retention Selectivity N = (gas, L, r c ) k = (T, d f, r c ) a = (T, phase) L = Length r c = column radius d f = film thickness T = temperature

Column Diameter - Theoretical Efficiency 5 m Total Plates I.D. (mm) N ~ 112,000 0.05 n/m 23,160 10 m N ~ 112,000 0.10 11,580 0.18 6,660 20 m N ~ 112,000 0.20 5830 0.25 4630 30 m N ~ 112,000 0.32 3660 0.45 2840 k = 5 0.53 2060

Column Diameter and Capacity I.D. (mm) Capacity (ng) 0.05 1-2 0.10 6-13 0.18 25-55 Like Polarity Phase/Solute 0.25 µm film thickness 0.20 35-70 0.25 80-160 0.32 110-220 0.45 600-800 0.53 1000-2000

Column Diameter and Carrier Gas Flow Lower flow rates: Smaller diameter columns Higher flow rates: Larger diameter columns Low flow rates : GC/MS High flow rates: Headspace, purge & trap

Diameter Summary To increase Efficiency Resolution Pressure Capacity Flow rate Diameter Smaller Smaller Smaller Larger Larger

Resolution N k Rs = 4 k 1 a 1 a Efficiency Retention Selectivity N = (gas, L, r c ) k = (T, d f, r c ) a = (T, phase) L = Length r c = column radius d f = film thickness T = temperature

Film Thickness and Retention: Isothermal Thickness (µm) Retention Change 0.10 0.40 0.25 1.00 1.0 4.00 3.0 12.0 5.0 20.0 Constant Diameter Normalized to 0.25 µm

Film Thickness and Resolution When solute k < 5 d f R (early eluters) or T When solute k > 5 (later eluters) d f or T R

Film Thickness and Bleed More stationary phase = More degradation products

Other Retention - Adsorption Analysis of Noble & Fixed Gases Using HP PLOT MoleSieve 2 4 5 Column: Carrier: Oven: Sample: HP-PLOT/MoleSieve 30 m x 0.53 mm x 50 m HP part no. 19095P-MS0 Helium, 4 ml/min 35 C (3 min) to 120 C (5 min) at 25 C/min 250 l, split (ratio 50:1) 6 1 3 1. Neon 2. Argon 3. Oxygen 4. Nitrogen 5. Krypton 6. Xenon 2 4 6 8 Time (min)

Film Thickness Summary To Increase Retention Resolution (k<5) Resolution (k>5) Capacity Bleed Inertness Efficiency Make Film Thicker Thicker Thinner Thicker Thicker Thicker Thinner

Phase Ratio ( ) Column Dimensions Phase Ratio β 30 m x.53 mm x 3.0 m 44 30 m x.32 mm x 1.8 m 44 β = r 2d f Kc = kβ (same phase ratio gives same retention)

Different Column I. D. Equal Phase Ratios Column: DB-624 30 m, 0.53 mm, 3 m Carrier: Oven: Helium, 40(cm/sec) 65 C Injection: Split Detector: FID Column : DB-624 30 m, 0.32 mm, 1.8 m 0 5 10 15 20 Time (min)

Resolution N k Rs = 4 k 1 a 1 a Efficiency Retention Selectivity N = (gas, L, r c ) k = (T, d f, r c ) a = (T, phase) L = Length r c = column radius d f = film thickness T = temperature

Column Length and Efficiency (Theoretical Plates) Length (m) N 15 69,450 30 138,900 60 277,800 0.25 mm ID n/m = 4630 (for k = 5)

Column Length and Resolution R a N a L Length X 4 = Resolution X 2 t a L Upside = Cut a bunch off during routine inlet maintenance and not lose a lot of Resolution

Column Length VS Resolution and Retention Time Isothermal R=0.84 2.29 min R=1.16 4.82 min R=1.68 8.73 min 15 m 30 m 60 m Double the plates, double the time but not double the the resolution

Column Length and Cost 15m 30m 60m $ $ $ $ $ $ $

Length Summary To Increase Efficiency Resolution Analysis Time Pressure Cost Length Longer Longer Longer Longer Longer

Resolution Impact of Efficiency, Selectivity and Retention on Resolution 7.00 6.00 Typical Method Development Parameters 5.00 4.00 3.00 Increase N Increase Alpha Increase k' 2.00 1.00 R s = N ½ /4 (a-1)/a k /(k +1) 0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Plates: 5000 10000 15000 20000 25000 Alpha: 1.10 1.35 1.60 1.85 2.1 k : 2.0 4.5 7.0 9.5 12.0

Conclusions Choose the stationary phase for the best separation in the least amount of time. (Use application specific columns when available) Temperature limits and inertness can play a role. Smaller diameter allows for shorter lengths but have less capacity. Choose film thickness for retention reasons. PLOT columns have strong retention, use where appropriate. Minimize length but have enough necessary for resolution.